Lentiviral Knockdown and Overexpression

Cisplatin and chemical reagents, including Tris, NaCl, SDS and DMSO, for molecular biology and buffer preparation were purchased from Sigma-Aldrich (St. Louis, MO). Lentivirus plasmids containing pLKO.1-shEZH2 (#1, TRCN0000040073; #2, TRCN0000040074; #3, TRCN0000040075; #4, TRCN0000040076; #5, TRCN0000040077), pLKO.1-shSUZ12 (#1, TRCN0000038724; #2, TRCN0000038725; #3, TRCN0000038726; #4, TRCN0000038727; #5, TRCN0000038728), and pLKO.1-shEED (#1, TRCN0000021204; #2, TRCN0000021205; #3, TRCN0000021206; #4, TRCN0000021207; #5, TRCN0000021208) were purchased from Thermo Scientific. The pCMV-HA vector (cat#631604) was purchased from CloneTech. The EZH2 expression construct pCMV-HA-hEZH2 (Addgene plasmid #24230), the luciferase reporter Puma-Luc (Addgene plasmid #16591), the pBV-Luc construct (Addgene plasmid #16539), pLKO.1-shGFP (Addgene plasmid #30323), the lentiviral packaging plasmid psPAX2 (Addgene plasmid #12260) and the envelope plasmid pMD2.G (Addgene plasmid #12259) were available on Addgene (Cambridge, MA). The Renilla luciferase reporter construct pRL-SV40 (Promega) was used as previously described [59 (link)].

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Liu H., Li W., Yu X., Gao F., Duan Z., Ma X., Tan S., Yuan Y., Liu L., Wang J., Zhou X, & Yang Y. (2016). EZH2-mediated Puma gene repression regulates non-small cell lung cancer cell proliferation and cisplatin-induced apoptosis. Oncotarget, 7(35), 56338-56354.

Publication 2016

Cisplatin NaCl SDS DMSO pCMV-HA-hEZH2 pLKO.1-shGFP psPAX2 pMD2.G pRL-SV40

Buffer Cisplatin Dmso Ezh2 Lentivirus Luciferase Nacl Plasmid Promega Puma Renilla luciferase Sv40 Tris Vector

Corresponding Organization :

Other organizations : Central South University, Second Xiangya Hospital of Central South University, Third Xiangya Hospital, Hunan Cancer Hospital, Albert Einstein College of Medicine

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Variable analysis

independent variables

ShRNA knockdown of EZH2, SUZ12, and EED using lentiviral plasmids
Overexpression of EZH2 using pCMV-HA-hEZH2 construct

dependent variables

Molecular and cellular outcomes (e.g., gene expression, cell behavior) resulting from the independent variable manipulations

control variables

Cisplatin and chemical reagents (Tris, NaCl, SDS, DMSO) for molecular biology and buffer preparation
PLKO.1-shGFP as a control shRNA
PRL-SV40 Renilla luciferase reporter construct as a control

positive controls

PCMV-HA-hEZH2 for EZH2 overexpression
Puma-Luc and pBV-Luc luciferase reporter constructs

negative controls

PLKO.1-shGFP as a control shRNA

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